Reduction/oxidation (redox) is an important mechanism of post-translational modification controlling a wide variety of cellular functions. Reactive oxygen species (ROS) produced from various sources, such as mitochondrial leakage and NAD(P)H oxidases (Noxs), oxidize signaling molecules and transcription factors. Noxs are major enzymes responsible for production of superoxide (O2-), a component of ROS, at various subcellular localizations. O2- inactivates iron-sulfur cluster containing enzymes, thereby liberating free iron, which in turn generates the highly reactive hydroxyl radical. O2- produced in cells is rapidly converted to H2O2, while O2- also reacts with NO to form ONOO- , all of which act as ROS. Although the cellular function of plasma-membrane associated Noxs, such as Nox1 and p91phox (Nox2), has been extensively characterized, that of Nox4, an isoform of Nox expressed in the heart, is not well understood. Although oxidative stress in the heart is increased by aging and stress and during cardiac failure, the mechanism and localization of ROS production are not well understood. Our preliminary studies suggest that expression of Nox4 in the heart is upregulated by aging and pressure overload. However, the contribution of Nox4 to the increased oxidative stress and the progression of cardiac aging and heart failure remains to be elucidated. Furthermore, molecular mechanisms (targets) regulated by Nox4 and their contributions to growth and death responses in the heart are currently unknown. Interestingly, our preliminary results suggest that inhibition of Nox4 is protective during pressure overload but is detrimental during ischemia/reperfusion. The overall hypothesis in this proposal is that although physiological levels of Nox4 mediate cellular functions essential for cell survival under certain stress conditions, upregulation of Nox4 in aging and failing hearts elicits detrimental effects, such as increased production of ROS and subsequent mitochondrial dysfunction. In particular, our specific hypotheses are:1. Upregulation of Nox4 during aging and pressure overload increases oxidative stress, apoptosis and cardiac dysfunction, and is thereby detrimental. 2. Nox4 is a critical mediator of mitochondrial oxidative stress and mitochondrial dysfunction during heart failure. 3. The presence of a physiological level of Nox4 protects the heart from ischemia/reperfusion (I/R) injury. Nox4 plays an essential role in upregulating HIF-11 during acute ischemia, which in turn mediates stimulation of glycolysis, thereby preventing cardiac myocyte death during I/R. We will address these issues using newly generated mouse models of both gain and loss of function of Nox4, including Nox4 transgenic and KO mice, proteomic analyses and integrated physiology studies. Our results will elucidate the role of Nox4 in mediating both physiological and pathological functions in the heart during aging and under stresses.